Longitudinal transmission of vasodilatory signals along microvascular arterioles (conducted vasodilation; CVD) underlies the coordination of blood flow within microvascular networks and is an important mechanism for integrating tissue perfusion and peripheral resistance. Little is known of how disease alters CVD within a microvascular network. Elevated blood concentration of homocysteine (hyperhomocysteinemia; HHcy) is a risk factor for several cardiovascular diseases. Though HHcy impairs microvascular reactivity to pharmacologic vasodilators, nothing is known of the functional deficits of microvascular blood flow control in HHcy. Based on pilot data and published literature, our working hypothesis is that HHcy impairs coordination of blood flow control in skeletal muscle by reducing cell-cell communication through gap junction and that this impairment can be reversed by exercise training. In Aim 1, we will explore a set of potential mechanisms for homocysteine-mediated dysfunction in conducted vasodilation and the connexins proteins that form gap junction communication pathways. Pilot data show that homocysteine disrupts CVD and increases serine phosphorylation of both connexin40 and connexin43, which are responsible for endothelial cell-cell (EC-EC) and endothelial-smooth muscle cell (EC-SMC; myoendothelial) communication. We develop the novel hypothesis that homocysteine activates metabotropic glutamate receptors on endothelial cells, mediating a G-protein cascade through phosphokinase C (PKC) and serine phosphorylation of these gap junction components. We will test this hypothesis using 1) a new co-culture system with primary EC and SMC from skeletal muscle microvessels, allowing for the first time the distinct isolation of myoendothelial junctions, and 2) isolated skeletal muscle arterioles in vitro. In Aim 2, we will evaluate the effects of exercise training on ascending vasodilation in HHcy. CVD that ascends from contracting skeletal muscle into respective feed arteries (ascending vasodilation; AVD) is an essential component of exercise hyperemia. Pilot data from mice with mild HHcy (deficiency in the homocysteine-metabolizing enzyme, CBS v wild type littermates) indicate an impaired capacity for AVD in response to muscle contraction. Electron microscopy shows significant changes in the morphology of myoendothelial junctions in skeletal muscle arterioles. Unfortunately, there are currently no efficacious clinical interventions for mild to moderate hyperhomocysteinemia. Using intravital microscopy of CBS and mice in vivo, we will test the hypothesis that exercise training can reverse homocysteine- mediated deficits in AVD and reverse structural deficits at the myoendothelial junction. Collectively, these experiments will provide new understanding of the microvascular dysfunction caused by HHcy, a risk factor for cardiovascular disease, venous thromboembolism, stroke, and Alzheimer disease. PUBLIC HEALTH RELEVANCE: The lining of the microvasculature, the endothelium, communicates nutrient needs of cells to upstream vessels via gap junctional coupling, thereby matching local metabolic needs with upstream vasodilation. Hyperhomocysteinemia, a risk factor for cardiovascular and neurocognitive disease, disrupts endothelial gap junctional communication. This project will 1) identify the mechanisms by which homocysteine disrupts this communication pathway and 2) elucidate the effects of exercise training on reversing the effects of hyperhomocysteinemia and restoring this vital communication pathway.